Electrowetting dispensing devices and related methods

ABSTRACT

A method for dispensing liquid for use in biological analysis may comprise positioning liquid to be dispensed via electrowetting. The positioning may comprise aligning the liquid with a plurality of predetermined locations. The method may further comprise dispensing the aligned liquid from the plurality of predetermined locations through a plurality of openings respectively aligned with the predetermined locations. The dispensing may be via electrowetting.

PRIORITY

This application is a continuation of U.S. application Ser. No.11/213,355, filed Aug. 26, 2005, which claims priority to U.S.Provisional Application No. 60/604,845 filed Aug. 26, 2004, entitled“Electro-wetting Loader,” which is incorporated by reference herein.

TECHNICAL FIELD

This invention relates to devices and related methods for handling anddispensing small volumes of liquids, such as, for example, in the fieldof microfluidics. In particular, the invention relates to devices andrelated methods utilizing electrowetting principles for handling anddispensing liquid for use in performing biological analysis (e.g.,testing, assays, and other similar procedures).

BACKGROUND

In the field of biological analysis and assays, small amounts of liquidmust often be dispensed to predetermined locations, for example, to aplurality of wells in titer plates, capillary tubes, and/or othersimilar test platforms, in order to perform various analyses (e.g.,testing, assays, and other procedures). Such dispensing is oftenautomated, as it is desirable to perform numerous tests at a relativelyhigh rate. To this end, it is also desirable to dispense a large numberof small volumes of liquid simultaneously. Further, it is desirable toprovide precise control over the amount of liquid dispensed, the timingof the dispensing, and/or the location of the dispensing in order toprevent wasting of materials and improve efficiency of the overalltesting procedure.

Conventional devices and methods for dispensing liquids, such as liquidsfor biological analysis, include the use of liquid handling robots andpipettes, which often are automatically controlled to dispense apredetermined amount of liquid into each well of a titer plate. Some ofthese liquid handling robot devices are moved to the appropriateposition corresponding to a predetermined dispensing location viamotors. Conventional techniques for dividing liquid into small amountsfor biological analysis include the use of capillary forces, vacuumforces, and centrifugal forces, for example. Some of these conventionalliquid handling devices aspirate and/or dispense liquid to some numberof wells at one time. In some cases, conventional liquid handlingdevices can only move liquid in or out of one well at a time. This typeof device typically is able to move about three axes so as to move overany well in a two-dimensional array of wells and to move toward and awayfrom a well. Other conventional liquid handling devices may have theability to fill multiple wells in a plate simultaneously, for exampleall wells in a plate, which may permit such devices to require less axesof motion to operate and to achieve faster operating rates.

Typically, such conventional dispensing devices are configured todispense liquid to, for example, a 96 well or 384 well titer plateconfiguration. To achieve faster sample testing rates (e.g., a higherthroughput of sample testing), it may be desirable to increase thenumber of testing locations (e.g., reservoirs, wells, capillary tubes,etc.) such that more samples can be dispensed onto a testing platformsimultaneously and analyzed. It may further be desirable to increase thenumber of reservoirs (e.g., wells) on a testing platform while keepingthe platforms' overall dimensions substantially the same. In otherwords, it may be desirable to increase the density of the testingreservoirs on the same testing platform area, such as, for example byincreasing the density of the reservoirs four-fold, eight-fold, and16-fold. In this way, new testing platforms with a larger number oftesting reservoirs could be retrofit with existing analytical systems.

In some conventional liquid dispensing devices, the size of theactuators (e.g., dispensers and/or aspirators) present a practical limitto the filling density these devices are able to achieve (e.g., thenumber of wells the liquid dispensing devices can fill simultaneouslyover a titer plate having a constant area). For example, forhigh-density spacing between wells (e.g., relatively small distancesbetween adjacent wells), the actuator of conventional dispensing devicesmay be larger than the well spacing, thereby preventing multipleactuators from addressing adjacent wells to dispense liquidsimultaneously into those wells.

Thus, it may be desirable to provide devices and methods for dispensingliquid for biological analysis that provide precise manipulation ofsmall volumes of liquid at relatively rapid rates. Further, it may bedesirable to provide relatively compact dispensing devices that canprovide both liquid handling (e.g., positioning) and dispensing to aplurality of locations on a testing platform. In addition, it may bedesirable to provide methods and devices that can be readilyincorporated into existing biological analysis systems (e.g.,workstations). For example, it may be desirable to provide a dispensingdevice and method that can dispense liquid to a testing platform havingsubstantially the same dimensions as conventional testing platformswhile increasing the number of locations for depositing liquid forperforming testing, for example increasing the number of locations(e.g., wells) to 96, 384, 768, 1536, 3072, 6144, 12,288, 24,576, etc. Inother words, it may be desirable to provide dispensing devices andmethods that permit higher density dispensing of liquid, including, forexample, ultra-high density dispensing applications, which may improvethe overall efficiency of biological analysis systems by increasing thenumber of tests that can be performed at a time. In providing methodsand devices that permit handling and dispensing of smaller volumes ofliquid at a higher density, it may further be desirable to minimizeevaporation of the liquid.

Yet further desirable features include providing dispensing devices andmethods that can minimize wasted liquid during dispensing, can divide anamount of supplied liquid into precise smaller amounts, and/or deliverthose precise amounts accurately to predetermined locations. It also maybe desirable to provide dispensing devices and methods that are capableof positioning and delivering smaller amounts of liquid thanconventional dispensing devices, for example on the order of a fewmicroliters and/or a few nanoliters.

Another desirable aspect includes providing dispensing devices andmethods capable of multi-plexing, i.e., handling and dispensingmultiple, differing types liquids, and capable of doing so with minimalrisk of cross-contamination of the differing types of liquid.

Further, it may be desirable to provide dispensing devices that arereusable for repeated handling and dispensing operations, and to providedispensing devices and methods that are robust, reliable, and/or reduceoverall costs of handling and dispensing operations.

SUMMARY

Dispensing devices and methods according to exemplary aspects of thepresent invention may satisfy one or more of the above-mentioneddesirable features. Other features and advantages will become apparentfrom the detailed description which follows.

In various applications relating to liquid handling, for example in thefield of microfluidics, electrowetting has been used to manipulateliquid behavior. As used herein, electrowetting involves the use of anelectric field to alter the wetting behavior of liquid relative to asurface so as to control the movement of the liquid. In other words,through the application of an electric potential, a liquid-solidinterface can be altered by controlling the wettability of the surface(e.g., effectively converting the surface in contact with the liquidfrom hydrophobic to hydrophilic or vice versa) to thereby controlmovement of a liquid on that surface. Thus, electrowetting can be usedto precisely divide and position liquid, without the need to utilizepumps, valves, channels, and/or other similar fluid handling mechanisms.

As an example, electrowetting may include sandwiching the liquid betweentwo plates and in contact with an insulated electrode. By applying anelectric field in a non-uniform manner so as to create a surface energygradient, a large number of small volumes of liquid (e.g., droplets,beads, cells, or other small volumes) can be independently manipulatedunder direct electrical control and without the use of pumps, valves, orfixed channels. Moreover, as will be explained in the description whichfollows, electrowetting may be used to achieve relatively precisemovement of liquid on a surface in relatively larger amounts, e.g.,without necessarily requiring first dividing the liquid into droplets orthe like.

For further information on electrowetting and exemplary deviceconfiguration and applications for implementing electrowetting,reference is made to U.S. Pat. No. 6,565,727 B1, which issued on May 20,2003; U.S. Publication No. 2003/0205632 A1, which published on Nov. 6,2003, and U.S. Publication No. 2003/0006140 A1, which published on Jan.9, 2003, the entire contents of each of which are incorporated byreference herein. To the extent that any conflict may exist between theteachings of the above-cited patent documents and this application, theteachings of this application should apply.

In accordance with exemplary aspects of the invention, the use ofelectrowetting in the field of liquid handling for biological analysismay provide relatively accurate and fast manipulation of a large numberof small volumes of liquid. As discussed above, there is a need fordispensing liquid used in biological analysis (e.g., assays, testing,and other related procedures) into numerous small reservoirs, such aswells in titer plates, for example, with a compact device (e.g., loader)that provides both liquid handling (e.g., positioning) and dispensing.Such a device may replace a liquid handling robot or be incorporatedwithin a biological analysis workstation. The loading configuration ofthe dispensed liquid may be programmed by computer, e.g. a 96-, 384-,768-, 1536-, 3072-, 6144-, 12,288-, or 24,576-well format. The amount ofliquid dispensed may include drops, cells, beads, or other amounts, inan exact number (e.g., the amount of liquid dispensed may becontrolled). Moreover, the precise locations to which the liquid isdispensed may be controlled.

According to exemplary aspects of the invention, supplied liquid may bedivided into smaller, precise portions (e.g., volumes) and dispensed.The dispensed volumes can be very small, for example, on the order of afew microliters and/or a few nanoliters. By way of example only, thedispensed volumes may range from about 0.01 microliters to about 100microliters, for example from about 0.01 microliters to about 5microliters. In an exemplary aspect, the volume may be about 1microliter. A wide range of volumes are envisioned depending on theparticular application. Further, according to another exemplary aspect,a dispensing device may handle multiple, differing types of liquidsamples (e.g., multi-plexing). For example, a dispensing deviceaccording to aspects of the invention may have more than one sampleinput port such that differing samples can be input to the device viadiffering input ports, moved and positioned in a segregated fashionthroughout the dispensing device, and then distributed to differinglocations of a testing platform. In an alternative example, differingliquids may be input via differing ports and mixed together within thedispensing device and then dispensed to the testing platform. Since thedispensing devices and methods according to aspects of the invention maybe programmable, the chance of cross-contamination when performingmulti-plexing procedures may be reduced.

A dispensing device (e.g., loader) according to exemplary aspects of theinvention may replace sample-positioning motors by manipulating dropsinto view. For example, if an operator is looking through a microscopeat a marked electrode in a transparent dispensing device, the operatorcan manipulate a sample drop into the microscope view without having tofirst find the location of the sample in the dispensing device prior tolooking through the microscope.

Assuming the input and dispensed volumes of liquid are the same,dispensing devices and methods according to aspects of the presentinvention may minimize wasted liquid as compared to conventional fluiddispensing devices. Further, according to yet another exemplary aspect,evaporation of liquid may be minimized when dividing liquid intorelatively small volumes and dispensing those small volumes torelatively high density testing platform formats.

According to yet an additional exemplary aspect, when dispensing liquidto capillaries, reagent costs may be reduced, for example by orders ofmagnitude. At least some of the dispensing devices and methods accordingto aspects of the invention result in a robust and reliable handling anddispensing operation. Moreover, in an exemplary aspect, the dispensingdevices may be reusable for repeated dispensing of samples.

According to an exemplary aspect of the invention, as embodied andbroadly described herein, the invention may include a method fordispensing liquid for use in biological analysis comprising positioningliquid to be dispensed via electrowetting. The positioning may comprisealigning the liquid with a plurality of predetermined locations. Themethod may further comprise dispensing the aligned liquid from theplurality of predetermined locations through a plurality of openingsrespectively aligned with the predetermined locations.

According to yet another exemplary aspect, the invention may include amethod for dispensing liquid for use in biological analysis comprisingsupplying a liquid to be dispensed to a housing comprising an interiorsurface and altering a wettability of the interior surface so as todivide the liquid in the housing into a plurality of individual portionsof liquid and to move the plurality of individual portions of liquid toa plurality of respective predetermined locations. The method mayfurther comprise dispensing the plurality of individual portions ofliquid from the plurality of predetermined locations through a pluralityof openings respectively aligned with the plurality of predeterminedlocations.

In the following description, certain aspects and embodiments willbecome evident. It should be understood that the invention, in itsbroadest sense, could be practiced without having one or more featuresof these aspects and embodiments. It should be understood that theseaspects and embodiments are merely exemplary and explanatory and are notrestrictive of the invention.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate embodiments of the invention andtogether with the description, serve to explain certain principles. Inthe drawings:

FIG. 1 is a schematic, perspective view of an exemplary embodiment of adispensing device configured for dispensing liquid to a titer plateaccording to an aspect of the invention;

FIGS. 2A-2D illustrate a schematic, partial cross-section of thedispensing device of FIG. 1 and various exemplary steps for positioningand dispensing a liquid according to aspects of the invention;

FIGS. 3A-3D schematically illustrate exemplary steps for positioningliquid in a dispensing device according to aspects of the invention;

FIGS. 4A and 4B are schematic partial views of an exemplary embodimentfor dispensing liquid from the dispensing device of FIG. 1 into wells ina titer plate;

FIGS. 5A and 5B are schematic partial views of another exemplaryembodiment for dispensing liquid from the dispensing device of FIG. 1into wells in a titer plate;

FIGS. 6A and 6B are schematic partial views of yet another exemplaryembodiment for dispensing liquid from the dispensing device of FIG. 1into wells in a titer plate;

FIG. 7 is a schematic partial view of an exemplary embodiment fordispensing liquid from the dispensing device of FIG. 1 to capillarytubes;

FIG. 8 is a schematic, perspective view of an exemplary embodiment of abiological analysis workstation;

FIG. 9 is a schematic, perspective view of another exemplary embodimentof a dispensing device according to an aspect of the invention;

FIGS. 10A-10D are schematic side views of another exemplary embodimentof a dispensing device and various exemplary steps for positioningliquid;

FIGS. 11 and 11A are a plan view of an exemplary embodiment of a routingplate in accordance with an aspect of the invention;

FIGS. 12A-12G schematically illustrate various exemplary steps forpositioning and dividing liquid along the routing plate of FIG. 11;

FIGS. 13A and 13B are partial, schematic perspective views of anexemplary embodiment of a nozzle plate according to an aspect of theinvention; and

FIG. 14 is a schematic, perspective view of another exemplary embodimentof a biological analysis workstation.

DETAILED DESCRIPTION

FIG. 1 shows an exemplary embodiment of a dispensing device according toaspect of the invention. The dispensing device 20 shown in FIG. 1 isreferred to herein as an electrowetting loader (EWL). In the exemplaryembodiment of FIG. 1, the EWL 20 is shown in conjunction with a titerplate 100 comprising a plurality of wells 150 to which the EWL 20 isconfigured to dispense small portions (e.g., volumes) of liquid forbiological analysis. The EWL 20 comprises a first substrate 25. Aplurality of electrodes 45, which may be in the form of electrical pads,for example, are embedded in the first substrate 25. As shown in FIG. 1,the electrical pads 45 may be arranged so as to form a two-dimensionalarray of rows 16 and columns 17. Each pad 45 in the substrate 25 can beindependently charged positive or negative relative to a power sourceprovided in an electrical controller 30. For example, each pad 45 may beseparately wired to the controller 30.

A thin, hydrophobic insulator layer 24 may cover the electrical pads 45,as shown in FIGS. 2A-2D. An input port 40, which may be in the form of ahydrophilic through-hole, for example, in the substrate 25 permitsloading of a first amount of liquid, for example, several milliliters,into the EWL 20. By way of example, to fill a 1536-well format titerplate with 4 microliters per well, at least 6.144 milliliters of liquidmay be supplied from the input port 40 to the dispensing device 20. Aswill be explained in more detail below, the electrical pads 45 may bearranged in columns 17 and rows 16 so as to separate, position, anddispense a plurality of individual exact (e.g., predetermined) volumesof liquid supplied from the input port 40 and move those individualvolumes, which may be in the form of droplets, for example, in any twodimensional movement within the EWL 20 that is desired.

The EWL 20 further comprises a second substrate 60 disposedsubstantially opposite the first substrate 25 and separated from thefirst substrate 25 by a small distance (for example, ranging from about0.1 millimeters to about 10 millimeters, such as, for example, about 1millimeter.) A seal 65, such as, for example, adhesive (e.g.,double-sided tape), a polymer gasket, metallic seals, or other similarseals, may be provided along the edges of the substrates 25, 60, asshown in FIGS. 2A-2D, to hold the substrates 25, 60 spaced apart fromone another. The seal 65 may define, in conjunction with the substrates25, 60, a chamber 70 (e.g., cavity) configured to receive liquid fromthe input port 40 and to move and position the liquid inside the EWL 20.The second substrate 60 may have one relatively large embedded electrode75 that can be independently charged positive or negative relative tothe power source associated with the electrical controller 30. That is,the electrode 75, like the electrodes 45, may also be separately wiredto the controller 30. As shown in FIGS. 2A-2D, a thin, hydrophobicinsulator layer 74, which may be similar to insulator layer 24, maycover the electrode 75.

Thus, the first substrate 25, second substrate 60, and seal 65 togethermay form a housing having an interior surface, which in the exemplaryembodiment of FIGS. 1 and 2, includes the surfaces of insulator layers24, 74 facing chamber 70. At least one interior surface portion of thehousing may be configured so as to be electrically conductive. Forexample, at least one interior surface portion may comprise a dielectricmaterial covering an electrical conductor, such as, for example, theinsulator layers 24, 74 covering the electrodes 45, 75 in the embodimentof FIGS. 1 and 2.

The second substrate 60 may define at least one opening therethrough,e.g., a plurality of openings 50. The openings 50 may be in the form ofsmall, hydrophilic through-holes (e.g., exit ports) that can be arrangedand configured so as to align with predetermined locations (e.g.,reservoirs) on a testing platform. For example, as shown in FIG. 1, theopenings 50 may be configured and arranged so as to align with wells 150in a titer plate 100. That is, the number and arrangement of theopenings 50 may have substantially the same number and arrangement of aparticular titer plate format, e.g., 96-, 384-, 768-, 1536-, 3072-,6144-, 12,288-, or 24,576-well format.

According to an exemplary aspect, the openings 50 may be lined with(e.g., coated with a layer of) a material that exhibits hydrophiliccharacteristics. In an alternative embodiment, the openings 50 may beconfigured so as to be capable of exhibiting hydrophilic characteristicsupon application of an electric field thereto. In other words, in amanner similar to that which will be described below with reference tothe nozzle plate of FIGS. 9, 10 and 13, the openings 50 may beconfigured so as to be capable of drawing liquid to be dispensed intothe openings 50 via electrowetting. In either case, during filling, theopenings 50 may exhibit hydrophilic characteristics such that the liquidmoves from predetermined locations within the chamber 70 into theopenings 50. In the example wherein the openings are configured tobecome hydrophilic, prior to filling the openings with liquid, theopenings may exhibit hydrophobic characteristics.

FIGS. 2A-2D schematically illustrate a side, cross-sectional view of theEWL of FIG. 1 and illustrate how the EWL 20 aliquots a portion (e.g.,first amount) of the liquid supplied to the housing (e.g., chamber 70)from the input port 40 into small portions (e.g., droplets) ofpredetermined volume less than the first amount and moves those dropletsthrough the chamber 70 via electrowetting so as to ultimately bedispensed through one or more openings 50. For ease of reference, only aportion of the EWL 20 is illustrated to show how the electric charge ofthe electrodes 45, 75 can be controlled (e.g., via controller 30) so asto move liquid within the chamber in a single direction to be positionedin alignment with an opening 50 for dispensing therethrough. FIG. 3,discussed below, schematically illustrates how liquid can be moved intwo dimensions within the chamber 70 and aligned with predeterminedlocations by arranging the electrical pads 45 in a two-dimensional arrayof rows and columns and controlling the charge of the pads 45 in amanner similar to that described with reference to FIG. 2.

Referring to FIG. 2A, a relatively large volume of liquid 200 (on theorder of several milliliters) is supplied to the input port 40. By wayof example, the input volume of liquid may range from about 0.1microliter to about 10 milliliters. The liquid fills the hydrophilicport 40 but is prevented from moving (e.g., spreading) into the chamber70 of the EWL 20 by the hydrophobic insulator 24 provided on the surfaceof the substrate 25 facing the chamber 70. In other words, because theinput port 40 extends into the chamber, liquid is able to fill thehydrophilic port 40 so as to touch the insulator layer 74 of the secondsubstrate 60. However, portions of the hydrophobic surfaces 24, 74adjacent the input port 40 act to repel the liquid 200, therebypreventing the liquid 200 from moving away from the input port 40 andfurther into the chamber 70.

As shown in FIG. 2B, once it is desired to begin moving the liquid fromthe input port 40 throughout the chamber 70, power may be supplied fromthe controller 30 so that the electrode 75 of the second substrate 60may be negatively charged while the first two electrical pads 45adjacent to the input port 40 (e.g., the pads occupying positions i andii labeled in FIG. 2) are positively charged. The relative charges ofthe electrodes are indicated by +/− in FIGS. 2A-2D. By altering theelectric potential in this manner, a charge builds up at the insulatorsurface 24 but not on the insulator surface 74 of the second substrate60. Supplying the pads 45 with a positive electrical charge, relative tothe negative electrical charge of the electrode 75, effectively convertsthe pads 45 into electrical capacitors and causes the wetting angle atthe surface portion of the insulator 24 facing the chamber 70 andpositioned below the positively charged pads 45 (e.g., the insulator24/liquid 200 interface) to change from hydrophobic to hydrophilic. Inother words, by controlling the electric charge, the wettability of thesurface portion in contact with the liquid is altered so as to controlmovement of the liquid. The now converted hydrophilic surfacecorresponding to the location of those positively charged pads, e.g., atposition i and ii in FIG. 2B, draws liquid 200 from the port 40. Theliquid 200, however, does not travel further to the pad 45 occupyingposition iii because that pad 45 is negatively charged in FIG. 2B.

FIG. 2C demonstrates an example of how liquid can be drawn further intothe chamber 70 and cut into an individual portion (e.g., an independentdroplet) of liquid. As shown and explained above with reference to FIG.2B, the first and second pads 45 adjacent to the port 40 (e.g., pads 45occupying positions i and ii, respectively) are positively charged suchthat liquid wets the surface below them. To divide the liquid 200 in thechamber 70 into a smaller portion, such as, for example, droplet 201,the pad 45 occupying position ii is positively charged and pad 45occupying position i as well as pads 45 on both sides of the pad 45occupying position i (perpendicular to the plane of FIG. 2C and notshown) are negatively charged. Controlling the electric charges in thismanner creates a force that squeezes a portion of the liquid 200 into anindependent droplet 201 resting on the surface corresponding to thepositively charged pad 45 occupying position ii. In other words, asexplained above, the negatively charged pads essentially return theportion of the surface of the insulator 24 facing the chamber 70 andpositioned in alignment with those negatively charged pads to theoriginal hydrophobic state, altering the wettability of the surfaceportion (e.g., the liquid/surface contact angle) from its previous stateand thereby repelling the liquid away from it and toward the surfaceportion of the insulator 24 positioned in alignment with the positivelycharged pad 45 occupying position ii.

Once a droplet 201 of the liquid is cut, further controlling the chargeof pads 45 at positions surrounding the droplet can move a drop alongcolumns and rows substantially from pad to pad. In an exemplary aspect,the droplet volume may be designed to slightly overflow onto all padsthat occupy positions adjacent to the pad at which a droplet is located.To move the individual droplet from one pad (the resting pad) to anotheradjacent pad, the adjacent pad to which it is desired to move thedroplet is made positively charged such that the insulator surfaceportion at a location corresponding to that pad becomes hydrophilic andattracts the droplet. At the same time, the resting pad is madenegatively charged such that the insulator surface portion at thelocation corresponding to that pad becomes hydrophobic, repelling thedroplet to thereby push the droplet off the resting pad and onto theadjacent pad. This procedure of controlling the charge of the electricalpads 45 can move a droplet indefinitely along a row of pads, includingturning corners so as to permit the droplet to travel along a column ofpads.

FIG. 2D illustrates an example of how a droplet 201 may be drawn into anopening 50 (e.g., throughhole) in the second substrate 60. Hydrophilicopenings 50 in the second substrate 60 may be disposed such that theyare substantially opposite to at least some of the electrical pads 45 inthe first substrate 25. By controlling the charges of the electricalpads 45, including an electrical pad 45 aligned with the opening 50(e.g., the pad occupying position v in FIG. 2D), a droplet 201 can beattracted to the pad 45 when the pad 45 is positively charged andsubsequently drawn into the opening 50 by capillary force so as to fillthe opening 50. If the outer surface (the surface facing away fromchamber 70) of the second substrate 60 is hydrophobic, and the opening50 is appropriately sized and configured, the droplet 201 will not flowout of the opening 50 absent some additional force acting on the droplet201 to express it from the opening 50. Various exemplary devices andmethods for dispensing droplets from the openings 50 will be explainedbelow with reference to FIGS. 4-7.

FIG. 3 shows an exemplary embodiment of how multiple droplets 201 can becut and moved through the EWL chamber 70 in two dimensions such that themultiple droplets 201 can fill respective multiple openings 50 in achosen format. As with FIG. 2, only a small portion of the electricalpad arrangement embedded in the first substrate 25 is illustrated forease of reference. It should be understood that the number of electrodesand arrangement thereof can vary depending on such factors as thedesired movement of the individual portions of liquid, the volume of theindividual portions of liquid to be dispensed, the desired positioningof the individual portions of liquid prior to dispensing, thearrangement and configuration of the openings in the second substrate,the testing platform format, and/or other factors relating to thedesired application in which the dispensing device will be utilized.

In various other embodiments, and in accordance with aspects of theinvention, the electrodes may be replaced with photosensitive materialpermitting control over the liquid (e.g., including over droplets) byincident light as described in U.S. Application Publication No.2003/0224528 A1, which published on Dec. 4, 2003, the entire content ofwhich is incorporated by reference herein. To the extent that anyconflict may exist between the teachings of the above-cited publishedapplication and this application, the teachings of this applicationshould apply.

With reference to FIG. 3A, droplets of liquid 201 are first cut from theinput port 40 as described with reference to FIG. 2B above. For theexample shown in FIG. 3, four droplets 201 of liquid will be cut andeventually moved throughout the chamber of the EWL 20 to four respectiveopenings 50 in the second substrate. The droplets 201 are first movedalong a first column 17 a of electrodes (e.g., electrical pads 45) thataligns with the input port 40. The movement of the droplets 201 alongthe electrical pads 45 in the column 17 a can be achieved by controllingthe relative electrical charges of the pads 45, as described withreference to FIG. 2C above. As shown in FIG. 3A, the droplets 201 arebrought to respective positions along the column of electrical pads atwhich rows 16 a, 16 b of electrical pads 45 intersect the column 17 a.

In FIG. 3B, the droplets 201 are directed at a right angle onto theintersecting electrode rows 16 a, 16 b. The droplets 201 are moved alongthe rows 16 a, 16 b and positioned again at row-column intersections.That is, the droplets 201 are moved respectively to electrical pads 45in rows 16 a, 16 b that intersect with additional (e.g., secondary)columns 17 b, 17 c, 17 d, 17 e of electrical pads 45. Each of thesecolumns 17 b, 17 c, 17 d, 17 e ends with an electrical pad 45 beingpositioned substantially in alignment with openings 50 in the secondsubstrate 60.

FIG. 3C illustrates an example of how the droplets 201 may be movedalong secondary columns 17 b, 17 c, 17 d, 17 e into positions (e.g., topredetermined locations) that are respectively aligned with the holes50. FIG. 3D illustrates how the droplets 201, once positionedappropriately, may be drawn into the holes 50, such as, for example, bycapillary force as explained with reference to FIG. 2 above.

Once positioned within the EWL 20 and drawn into the holes 50, theindividual droplets 201 are ready to be dispensed from the EWL 20 to aplurality of predetermined locations on a testing platform, such aswells in a titer plate, capillary tubes, or locations on a microscopeslide, for example. FIGS. 4-7 illustrate exemplary embodiments forvarious techniques to achieve the dispensing of the droplets 201 fromholes 50 to a testing platform so that biological analysis procedurescan be performed.

FIG. 4 shows an exemplary embodiment of a technique for dispensing thedroplets 201 from the EWL 20 to a plurality of wells 150 in a titerplate 100 using centrifuging. In the schematic illustration of FIG. 4A,a partial, side cross-sectional view of the EWL 20 is shown withdroplets 201 drawn into the holes 50 and held therein via capillarity.In the exemplary embodiment of FIG. 4A, the second substrate 60 of theEWL 20 is clamped into contact with the well-side of the titer plate 100via a clamping mechanism 160. Suitable clamping mechanisms for clampingthe EWL 20 and titer plate 100 together may include, but are not limitedto, the use of snap fit mechanisms, elastic bands, springs, gravity,screws, bolts, and/or mechanical pressure, for example. The clamping mayoccur prior to the start of the loading/positioning of the liquid withinthe EWL 20. The centers of the EWL holes 50 may be substantially alignedwith the centers of the wells 150. After electrowetting is utilized tofill the holes 50 with liquid droplets 201, for example, as describedabove with reference to FIGS. 2 and 3, the titer plate 100 and EWL 20are placed in a centrifuge 400 and centrifuged together such that thedroplets 201 are forced from the holes 50 and into the respectivelyaligned wells 150.

Once the droplets 201 are dispensed to the wells 150, the EWL 20 may beunclamped and removed from contact with the titer plate 100, as shown inFIG. 4B. The titer plate 100 may then be brought to a station or thelike to perform biological analysis on the liquid 201 in the wells 150.

Though FIG. 4 illustrates an exemplary embodiment of dispensing droplets201 from the EWL 20 into wells of a titer plate, it should be understoodthat a similar method could be used to dispense droplets via centrifugeonto predetermined locations of a two-dimensional surface such as, forexample, a microscope slide.

FIG. 5 schematically illustrates another exemplary technique fordispensing droplets 201 from the EWL 20. The exemplary embodiment ofFIG. 5 demonstrates a technique whereby wells 150 in a titer plate 100are coated with a hydrophilic material 152 and used to draw the droplets201 into the wells 150 from the holes 50 in the EWL 20. As in FIG. 4A,the second substrate 60 of the EWL 20 may be clamped into contact withthe well-side of a titer plate 100 prior to the start of loading andpositioning of the liquid in the EWL 20. The centers of the EWL holes 50may be substantially aligned with the centers of the wells 150.Electrowetting may then be utilized to fill the holes 50 with thedroplets 201 of liquid.

In the exemplary technique of FIG. 5, the volume of each droplet 201 maybe selected so as to be greater than the volume of the hole 50 throughwhich the droplet 201 is dispensed such that the droplet 201 extends outof the hole 50 and forms a meniscus M, as shown in FIG. 5A, when it isheld in the hole 50 via capillarity. The hole 50 may be designed suchthat its diameter is slightly larger than the diameter of thecorresponding well 150 to which it is aligned. This configurationpermits the meniscus M to touch the lateral wall of the well 150 whenthe droplet 201 passes through the hole 50. The hydrophilic coating 152provided on the inside surface of the well 150 may then attract thedroplet 201, drawing the droplet 201 from the hole 50 into the well 150.According to an exemplary aspect, only the inside of the well wall ismade hydrophilic such that the droplet is attracted into the well ratherthan on the top of the plate, which may be hydrophobic.

Examples of suitable hydrophilic materials that could be used to coatthe inside of the wells 150 include cations, anions, polyethyleneoxides, sugars, polyacrylamides, surfactants, and other hydrophilicmaterials. By way of example, amphiphilic, di-block copolymers may beused to coat the inside of the wells 150. Aside from coating the wellswith a hydrophilic material, it should be understood that any techniquemay be utilized to provide the wells with a hydrophilic inner surface,such as for example, bonding a layer of such material to the well innersurface, forming the wells out of a hydrophilic material, and othertechniques.

Yet a further exemplary technique for dispensing the droplets from theEWL 20 to a testing platform includes the use of electrowetting. In theexemplary embodiment of FIG. 6, electrowetting is utilized to cause thedroplets 201 to be dispensed from their positions within respectiveholes 50 to a testing platform. Using electrowetting to move thedroplets from the EWL 20 to a testing platform may obviate the need forcentrifugal dispensing, which may cause relatively violent motions on amicro-scale and potentially result in cross-contamination between wells.Further, centrifugation may require additional hardware, e.g., acentrifuge, and additional steps, which may make automation moredifficult.

In the exemplary embodiment of FIG. 6, a titer plate 100 (e.g., amicrotiter plate) is substantially uniformly coated with a thin layer ofan electrically conductive material 155, for example an electricallyconductive metal, such as, for example, gold, aluminum, indium tinoxide, or other electrically conductive material. By way of example, thethickness of the metal layer may range from about 50 angstroms to a fewmicrometers. For example, the thickness may be about 500 nanometers. Themetal layer 155 may be deposited via a variety of known techniques,including but not limited to vapor deposition, spray coating,electroplating, chemical vapor deposition, sputtering, spin coating,emersion, and other deposition techniques, for example. The depositedlayer 155 may then be electrically isolated by providing a hydrophobiclayer 156 over the layer 155. The hydrophobic layer 156 may be made froma polymeric material, such as, for example, cyclic olefin polymer,polymethyl methacrylate (PMMA), or Teflon-AF™, and may be applied by anysuitable technique, such as, for example, spray coating, spin coating,dip coating, in situ polymerization, and/or other coating techniques.The thickness of the hydrophobic layer may range from about 0.1micrometers to several micrometers, and may be, for example, about 0.1micrometers. According to an exemplary aspect, the testing platform(e.g., titer plate, card, etc.) may be made of a hydrophobic materialsuch that masking it from a hydrophilic coating may suffice for ahydrophobic barrier. According to some aspects, the hydrophobic layermay have a thickness on the order of approximately 500 nm.

As with the exemplary techniques of FIGS. 4 and 5, the EWL 20 may beclamped via a clamping mechanism 160 to the well side of a titer plate100, as shown in FIG. 6A, prior to loading and positioning the droplets201 within the EWL 20. The centers of the EWL holes 50 may besubstantially aligned with the centers of the wells 150. Electrowettingmay then be utilized to fill the holes 50 with the droplets 201 ofliquid, as described above.

When it is desired to dispense the droplets 201 from the holes 50 to thewells 150, movement of the droplets 201 may be actuated by applying anegative potential to both the first and second EWL substrates 25, 60(shown in FIGS. 2D and 6A) and applying a positive potential to themetal layer 155 deposited on the titer plate 100. This renders the EWLsubstrates 25, 60 hydrophobic and the titer plate layer 156 hydrophilic,resulting in the droplets 201 being attracted to the titer plate 100 andbeing drawn into the wells 150 by capillary wicking when the droplets201 touch the wells 150. Thus, in the exemplary embodiment of FIG. 6,electrowetting is used to dispense the liquid droplets 201 from the EWL20.

Numerous procedures exist in micromachining literature for coatingpolymer substrates, such as a titer plate, with metals. It is envisionedthat any of these methods may be used to coat the titer plate (or othertesting platform) with a metal layer as desired. One exemplary method offorming a metal layer on such a substrate is by using physical vapordeposition (PVD). Typically, the polymer substrate is activated bytreatment with oxygen plasma (corona discharge), which may be followedby PVD of, for example, chromium, tungsten, or titanium adhesion layers,which in turn may be followed by PVD of, for example, gold. All of theseoperations may be performed under high vacuum environments to preventoxidation of adhesion layers. Alternate methods for depositing metallayers on a substrate, such as a polymer substrate, for example a titerplate, include metal deposition by sputtering, electro-deposition,electro-chemical-deposition, electro-less deposition, and otherdeposition techniques. As discussed above, the hydrophobic layer can beapplied to the metal surface by spin coating, dip coating, in situpolymerization, spray coating, and/or other coating techniques.

Yet a further exemplary technique for dispensing the droplets 201 fromthe EWL 20 after they have been positioned as desired and moved into theholes 50 of the second substrate 60 is illustrated in FIG. 7. FIG. 7illustrates a technique (e.g., electrophoresis) whereby the droplets 201may be drawn into capillary tubes 170 (e.g., into reservoirs defined bythe capillary tubes 170) by capillary force attraction of the tube,which may be lined with a hydrophilic material or otherwise providedwith a hydrophilic inner surface. As shown in FIG. 7, in an exemplaryaspect, a plurality of capillary tubes 170, which may be held in asupport 175, are filled with a matrix 171 that may comprise a porouspolymer, for example, which permits movement of nucleic acid 300 thatmay be contained in the droplets 201. In an exemplary aspect the tubes170 may be made of glass, for example, or other naturally hydrophilicmaterial, such that the drops 201 are attracted to the tip of thecapillaries 170 placed adjacent to the drops 201 when the EWL 20 isclamped to the capillary tube support 175.

Conventional capillary electrophoresis devices use electro-injection ofDNA to introduce a plug of DNA into the small diameter of a glasscapillary tube. To initiate electro-injection, the loading end of acapillary is immersed into a liquid sample containing nucleic acid. Thesample is in contact with an electrode and the matrix on the distal endof the capillary is in contact with another electrode. A voltage may beapplied, for example, about 1500 volts, between the electrodes such thatthe negatively charged nucleic acid is attracted to the distal end ofthe capillary tube.

Electro-injection may concentrate most of the nucleic acid from thesample liquid into a small band inside the tip of the capillary tube.After electro-injection, capillary electrophoresis can begin, which usesmuch higher voltages applied between the electrodes, for example, about30,000 volts. This process can be very inefficient because the DNAsample volume into which the capillary tube is inserted typically rangesfrom approximately 5 microliters to approximately 20 microliters.However, only about 0.1 microliter of that volume is electro-injectedinto the capillary tube, i.e. about 99% of the DNA sample is wasted.This waste is a result of the need for high resolution ofelectrophoresis separation of small nucleic acids from larger ones. Ifthe electro-injection spans over too long of a time period (e.g., overabout 20 seconds), the nucleic acid may accumulate in a relatively longsection (band) of the capillary tube, which may result in poorresolution at the detection end of the capillary tube. On the otherhand, if the electro-injection time period is short (e.g., about 20seconds or less), the acid may accumulate in a band that is relativelyshort, resulting in a relatively high resolution at the detection end.However, for short injection times, nucleic acid is collected from arelatively small volume of sample, typically about 0.1 microliters, forexample. If the total sample volume ranges from about 5 microliters toabout 20 microliters, the injection efficiency is very low.

Using the devices and methods according to aspects of the invention, a0.1 microliter volume of DNA could be divided via electro-wetting, forexample via the exemplary embodiments described herein, and delivered tothe capillary tube. In contrast to the conventional approaches,therefore, the sample volume is approximately the same as the injectionvolume such that wasted sample liquid may be substantially eliminatedand reagent costs may be reduced by a factor of almost 100.

According the exemplary embodiment of FIG. 7, the electrode 75 in thesecond substrate 60 of the EWL 20 may be the electrode utilized forelectro-injection. That is, the droplets 201 may be placed in electricalcontact with electrode 75 in the second substrate 60 and in electricalcontact with distal electrodes 172 of the capillary tubes 170 via theelectrically conductive matrix 171. Thus, electro-injection may beimplemented using a small liquid volume (e.g., droplet 201) of an amount(e.g., about 0.1 microliter) selected so as to match the volumetypically injected into a capillary tube using conventional techniques.In this way, using some of the devices and methods according to aspectsof the invention may result in potentially no loss of nucleic acidduring an electro-injection process.

As exemplified in FIGS. 4-7, according to aspects of the invention,dispensing the liquid droplets 201 aligned with the predeterminedlocations may include moving the liquid in a direction that issubstantially nonparallel to a plane defined by the two-dimensionalmovement of the liquid through the EWL 20. For example, in theabove-described embodiments, during dispensing, the liquid droplets 201move in a direction that is substantially perpendicular to the plane ofmovement of the liquid through the EWL 20 during the positioning andaligning of the liquid.

It is envisioned that the electrowetting dispensing devices according toaspects of the invention, such as the EWL 20 described above, could beconfigured as a stand-alone device, like a multi-tip pipettor, forexample, as a component of a device, like a liquid handling mechanismwithin an instrument, or as part of an overall system that includesfluid handling between differing devices. An example of such a system isillustrated in FIG. 8.

FIG. 8 shows an electrowetting dispensing device, such as the EWL 20,described in the embodiment of FIG. 1, incorporated in an automatedbiological analysis workstation 11. Workstation 11 can include acomputer 13 with a CRT monitor 15, a keyboard 18 and a mouse device 19.The computer 13, CRT display 15, mouse 19, and keyboard 18 may behardware components of a control system with an operator interface forprogramming the workstation to perform desired activities including, forexample, liquid handling by the EWL 20. Not illustrated in FIG. 8 is theconnection between the EWL 20 and the computer 13 or between the EWL 20and a controller, for example like controller 30 described in FIG. 1,for providing power to the electrodes of the EWL 20. However, thoseskilled in the art would understand that such connections are envisionedfor controlling the EWL 20 and are considered within the scope of theinvention.

By way of example only, the workstation could be configured to performbiological analysis comprising DNA sequence detection. To this end, theworkstation 11 may include a thermal cycling station 21 with anautomatically activated heated lid and real-time detection, a samplepreparation station 23, a sample storage station 28, a titer platestation 27 for automatically loading a titer tray into the thermalcycling station 21, a reagent storage 33, and wash stations 26 and 29.The various stations may be arranged on a work surface 22 with width D1and depth D2. A portion of the workstation at region 46 is shown cutaway in FIG. 8 to better illustrate the components in the work area. Forthermal cycling station 21 and titer plate station 27, various kinds ofdrives, such as, for example, motor and pneumatic drives can be used toselectively position the titer tray in and out of the thermal cyclerblock and to selectively close and open the thermal cycler lid.

In an exemplary aspect, a linear actuator system 31 may be utilized tomove the EWL 20 over the different stations for loading liquids fromcontainers at the various stations and dividing and dispensing smallervolumes of the liquids at the same or other stations. Liquids can alsobe pumped to the EWL by pumps 32 and 34. Linear actuator system 31 canbe configured to move in the direction of arrow 41 along track 35. TheEWL 20 can move in the direction of arrow 39 along arm 37. The EWL 20could be translated vertically in the direction of arrow 43 so that thelinear actuator system 31 can provide a cartesian XYZ placement of theEWL 20 to anywhere on the work surface 22 of workstation 11.

According to exemplary aspects, it is envisioned that the EWL 20 of theworkstation 11 could be rinsed and reused numerous times to performliquid positioning and/or dispensing operations.

FIGS. 9-13 illustrate further exemplary embodiments of dispensingdevices and methods for use in positioning and dispensing liquid to atesting platform for biological analysis. As shown in the partialperspective view of FIG. 9, a dispensing device 500 may comprise a firstsubstrate which may be in the form of a routing plate 501 (e.g., anelectrowetting loader card), for example, and a second substrate, whichmay be in the form of a nozzle plate 502, for example. As will beexplained in more detail below, the routing plate 501 may be configuredto employ electrowetting so as to take an input volume of liquid(including multiple differing types of liquid), divide the liquid intosmaller portions (e.g., volumes on the order of microvolumes ornanovolumes) of liquid, and position the liquid at predeterminedlocations along the plate 501. In an exemplary aspect, the routing plate501 is configured to align portions of liquid to be dispensed withpredetermined locations on the routing plate 501 that are substantiallyaligned with nozzles 520 on the nozzle plate 502.

The nozzle plate 502 may be configured so as to receive the smallerportions of liquid from the routing plate 501 via electrowetting and toexpress those smaller portions of liquid so as to dispense them to atesting platform for biological analysis. The testing platform mayinclude, for example, a microscope slide, a titer plate, capillarytubes, or other testing platforms. In an exemplary aspect, the nozzles520 of the nozzle plate may express the liquid so as to spot the liquidto a testing platform. In a further exemplary aspect, the nozzles 520may be configured so as to receive a small volume of liquid from therouting plate 501 and to hold that small volume until additional smallvolumes of liquid have been positioned by the routing plate. The liquidheld b the nozzles could then be expressed to a testing platform whenthe additional small volumes positioned in the routing plate are drawninto the nozzles.

It should be understood that for practical reasons the illustrations ofFIGS. 9-13 are partial views due to the relatively high density(including ultra-high density) applications that are envisioned (e.g.,for dispensing to 96-, 384-, 768-, 1536-, 3072-, 6144-, 12,288-, or24576- and higher number well formats in the case of dispensing to titerplates). By way of example, it may be possible to fabricate thedispensing device such that the nozzle plate includes as many as 400,000nozzles arrayed across the surface of an area approximating 3″×5″.

The dispensing device of FIG. 9 thus relies on electrowetting to divideand position an input volume of liquid via the routing plate 501 and todraw the smaller, divided portions of liquid into nozzles 520 of thenozzle plate 502 for expressing to a testing platform. The routing plate501 and nozzle plate 502 can be positioned relative to one another tocreate a chamber 570 for moving and dividing the liquid along therouting plate 501 via electrowetting. For example, according to someaspects, the routing plate 501 and nozzle plate 502 may be separatedfrom each other by a distance ranging from about 0.1 millimeters toabout 10 millimeters, for example the distance may be about 1millimeter. In an exemplary aspect, the chamber 570 formed between therouting plate 501 and nozzle plate 502 may have a volume so as tominimize evaporation of the liquid, especially in the case of smallerliquid portion/higher density applications (e.g., smaller volumes ofliquid being dispensed to a larger number of locations to a testingplatform).

The routing plate 501 and nozzle plate 502 may be sealed together in amanner similar to the first and second substrates 25, 60 described withreference to FIGS. 1 and 2. For example, sealing mechanisms, such as,for example, adhesive (e.g., double-sided tape), a polymer gasket,metallic seals, and/or other mechanisms capable of providing a seal, maybe provided along the edges of the plates and used to secure the platestogether in a spaced relationship along the edges of the plates.

Thus, according to an exemplary aspect, the routing plate 501, nozzleplate 502, and sealing mechanism may together form a housing having aninterior surface, for example, a surface facing chamber 570. At leastone interior surface portion of the housing may be configured so as tobe electrically conductive. For example, in an exemplary aspect, atleast one interior surface portion may comprise a dielectric materialcovering an electrical conductor.

According to an exemplary embodiment, all of the internal surfaces ofthe device 500, e.g., all of the surfaces coming into contact with theliquid, such as, for example, the inner surface of the routing plate501, the inner surface of the nozzle plate 502, and the inner surfacesof the nozzles 520 (which may define nozzle reservoirs), may beconfigured to be hydrophobic. The routing plate 501 and the nozzle plate502 may contain electrodes (e.g., electrodes 518 in routing plate 501,as shown in FIG. 9) or may otherwise be configured such that an electricpotential may be applied to each plate so as to selectively cause innersurface portions of the plates to become wettable (e.g., hydrophilic) byaltering (e.g., reducing) the contact angle between the liquid and theinner surface portion with which the liquid is in contact.

More specifically, in an exemplary aspect, an electric field associatedwith the routing plate 501 may be controlled so as to move an amount ofinput liquid along the routing plate and divide the liquid intoindividual portions to be dispensed. In other words, by controlling anelectric field, the wettability of various surface portions on therouting plate 501 may be altered so as to move, position, and/or divideliquid within the chamber 570. During the positioning/dividing stepstaking place along the routing plate 501, the nozzle plate 502 may bekept in an electrically charged state such that the inside surfaces ofthe nozzles 520 remain hydrophobic and liquid is repelled from enteringthe nozzles 520. Once the individual portions of liquid to be dispensedare positioned at predetermined locations along the routing plate 501,for example, in alignment with openings to the nozzles 520, the electricfield acting on the plates 501 and 502 may be controlled such that theindividual portions of liquid will be drawn from the predeterminedlocations along the routing plate 501 and into the respective nozzles520 via electrowetting and capillary action. By way of example, thepower and ground states of the plates 501 and 502 may be switched so asto cause the inner surfaces of the routing plate 501 in contact with theliquid to become hydrophobic and the inner surfaces of the nozzles 520to become hydrophilic, thereby repelling liquid from the routing plate501 and drawing the liquid into the nozzles 520 (e.g., into thereservoirs defined by the inner surfaces of the nozzles).

With reference to FIGS. 10A-10D, exemplary steps of dividing,positioning, and drawing liquid so as to be ready for dispensing usingan embodiment of a dispensing device 600 comprising a routing plate 601and a nozzle plate 602 are schematically illustrated. The views of FIGS.10A-10D are side, cross-sectional views of the dispensing device 600;the cross-section of the routing plate 601 shown in FIGS. 10A-10D istaken along the line 10-10 illustrated in FIG. 12A. Therefore, in theview shown in FIGS. 10A-10D, the direction of movement of the liquidalong the routing plate 601 is shown in only one dimension. It should beunderstood, however, that movement along the routing plate 601 is in twodimensions. Further, as will be seen, in an exemplary aspect, dispensingthrough the nozzles 620 may be in a direction substantially nonparallel(for example, perpendicular) to a plane defined by the two-dimensionalmovement of liquid along the routing plate 601. Details of how the inputliquid may be divided and moved along the routing plate 601 in anexemplary aspect are provided below in the description of FIGS. 11 and12.

In FIG. 10A, a liquid 700 to be dispensed is introduced through an inputport 640 provided in the dispensing device 600. The input port 640 maybe in flow communication with a main filler rail 615 provided in routingplate 601. In the cross-sectional views of FIGS. 10A-10D, the mainfiller rail 615 extends in a direction into the plane of the drawingsheet. The main filler rail 615 may be a surface underlain bylithographically formed electrodes, for example. In an exemplary aspect,the main filler rail may define a channel formed in the surface of therouting plate 601.

In a manner similar to that described with reference to FIG. 2, the mainfiller rail 615 can be configured such that it is capable of controllingthe movement of the input liquid from the input port 640 along thefiller rail 615 (e.g., in a direction into the plane of the drawingsheet) via electrowetting. For example, the routing plate 601 may beprovided with electrodes 618 that may be controlled as desired so as toposition and divide liquid supplied to the filler rail 615. In theexemplary step of FIG. 10A, the input liquid 700 is moved from the inputport 640 so as to substantially fill the main filler rail 615 that runsalong a side 603 of the routing plate 601.

Referring to FIG. 10B, once the main filler rail 615 has beensubstantially filled with liquid, smaller portions of the liquid in themain filler rail 615 may be drawn, again via electrowetting, into aplurality of side arms 617, which may comprise channels, defined by therouter plate 601. The side arms 617 extend in a direction substantiallyperpendicular to the main filler rail 615 so as to form a plurality ofsubstantially parallel rows. Due to the side, cross-sectional views ofFIGS. 10A-10D, only one such side arm 617 can be seen. As can be seen inthe exemplary embodiment of FIG. 10B, the side arms 617 of the routingplate 601 may be positioned such that each arm 617 is substantially inalignment with a row of nozzles 612 of the nozzle plate 602.

Once the side arms 617 of the routing plate 601 have been filled withliquid and the main filler rail 615 has been emptied, the electric fieldof the routing plate 601 may be controlled so as to divide the liquidfilling each of the side arms 617 into a plurality of individualportions of liquid 701 via electrowetting. FIG. 10C illustrates theliquid 700 in an arm 617 being divided into individual portions ofliquid 701 for dispensing. As can be seen in the exemplary step of FIG.10C, individual portions of liquid 701 may be formed at predeterminedlocations on the routing plate 601 that are in substantial alignmentwith respective nozzles 620 of the nozzle plate 602.

Once the liquid has been divided into the individual portions 701 to bedispensed and aligned with the nozzles 620, electrowetting may again beutilized to draw the individual portions of liquid 701 from theirrespective predetermined locations on the routing plate 601 and intorespective openings 622 leading to the nozzles 620. As shown in FIG.10D, by drawing the partitioned liquid 701 from the routing plate 601and into the nozzles 620 via electrowetting, the individual portions ofliquid 701 may be expressed through the nozzles 620 such that they forma droplet 702 or the like held by capillarity at the tip of each nozzle620. The dispensing device 600 may then be brought into alignment with atesting platform, such as, for example, a titer plate, a microscopeslide, or the like, and brought into contact with predeterminedlocations on such a platform to dispense (e.g., spot) the droplets 702onto the predetermined locations on the testing platform so thatbiological analysis can be performed. In an exemplary aspect, asillustrated in FIG. 10D, the individual droplets 701 of liquid aredispensed through the nozzles 620 in a direction that is substantiallyperpendicular to the two-dimensional plane of movement of the liquidalong the routing plate 601.

The volume of each droplet 702 that the nozzles 520 dispense may besubstantially the same as the volume of each respective individualportion of liquid 701 divided on the routing plate 601. Alternatively,it may be advantageous to provide the nozzle reservoir with a volumethat is about twice the volume of the droplet expressed from the nozzle.In this manner, the nozzle may function as a holding volume while asecond volume (e.g., individual portion) for dispensing is beingpositioned along the routing plate. When the second volume (e.g.,individual portion) is moved into the nozzles, the first volume (e.g.,individual portion) would be expressed from the nozzle.

FIG. 11 shows a schematic, top view of an exemplary embodiment of therouting plate 601 that may be used with a dispensing device as describedin FIGS. 9 and 10. FIG. 11A is a blown-up view of a section of therouting plate 601 of FIG. 11. The routing plate 601 may be substantiallyin the form of a card-like structure having a main filler rail 615extending along an edge 613 of the plate 601. A plurality of side arms617 may extend from the main filler rail 615, for example, in asubstantially perpendicular direction to the rail 615. As illustrated inFIGS. 10-12, the rail 615 and arms 617 may be defined as pathways on asurface of the plate 601, for example pathways defined by surfaceportions on the plate 601 underlain with electrodes. According to anexemplary aspect (not shown), the rail 615 and arms 617 may be formed aschannels in the plate 601. The arms 617 may be in flow communicationwith the rail 615. The main filler rail 815 may be configured so as tobe capable of containing a volume of liquid substantially equal to thetotal volume of liquid that the arms 617 are capable of containing. Byway of example, the main filler rail 615 may be configured to contain anamount of liquid ranging from about 0.1 microliter to about 10milliliters. According to one exemplary embodiment, to use the device600 to fill a 1536-well titer plate with 4 microliters of liquid perwell, the rail 615 should be configured so as to contain at least 6.144milliliters of liquid.

According to an exemplary aspect, and not illustrated in FIGS. 11 and11A, the routing plate 601 may comprise an array of electrodes that arepositioned in substantially along the filler rail 615 and arms 617 suchthat microfluidic pathways are formed along the filler rail 615 and arms617. In other words, by selectively activating the electrodes, surfaceportions on the routing plate 601 corresponding to those electrodes maybe altered so as to become more or less wettable (e.g., exhibithydrophobic characteristics or hydrophilic characteristics) so as toprovide precise control over the movement of liquid along the rail 615and the arms 617. In an exemplary aspect, the microfluidic pathways(e.g., the rail 615 and the arms 617) are underlain by lithographicallydefined arrays of electrodes.

Numerous techniques may be used to provide the electrodes in the routingplate and/or to otherwise configure the routing plate such that it iscapable of achieving electrowetting. For example, one such technique mayinclude implanting electrical pads in a substrate covered with ahydrophobic insulator layer, for example, in a manner similar to thatdescribed for first substrate 25 of FIG. 1.

Another technique may include fabricating the electrodes positionedunderneath the microfluidic pathways of the routing plate from asequence of steps that has been used for micro-electronic devices, suchas multi-chip modules, for example. For references which describe thesteps used for micro-electronic devices and multi-chip modules,reference may be made tohttp://www.cpmt.org/past_trans/cpmtb_toc_(—)9502.html, for example. Inaccordance with this technique, electrodes may be formed using a seriesof steps involving deposition of a suitable conductive material. Suchconductive materials may include, for example an indium/tin/oxide alloy,aluminum, gold, and/or other conductive materials. The conductivematerial may then be patterned with a subtractive process, such as, forexample, wet chemical etching or a dry process, such as plasma etching,for example. For example, a photo-masking process may be employed on topof a deposited conductive material, followed by a subtractive etchingprocess as described above, in order to remove unwanted conductivematerial and leave electrodes in place. After performing the subtractiveprocess to pattern the conductive material into an array of electrodes,a thin layer of a dielectric material may be applied. Suitabledielectric materials may include, for example, polymers, or oxidesand/or nitrides having high dielectric constants. The layer thicknessmay be a function of the dielectric constant of the material. Forexample, the higher the dielectric constant, the thinner the layer.According to an exemplary aspect, the dielectric material may be appliedin a layer having a thickness ranging from about 1 micrometer to about10 micrometers.

The layer of dielectric material may be applied so as to isolate theconductive patterns so as to form the desired electrode positioning(e.g., array) relative to the routing plate surface. First, theelectrode structure may be formed using the deposition and subtractiveprocesses described above. The dielectric material may then be appliedas a substantially continuous coating over the formed electrodes. Forexample, the dielectric material may be applied via spin coating,sputtering, chemical vapor deposition, plasma enhanced chemical vapordeposition, and/or other deposition techniques. The series of stepsdescribed above may be repeated as needed to achieve the desiredgranularity of the electrodes. The term granularity is used herein todescribe the size of the individual electrodes. For example, a highgranularity refers to a relatively high number of individual electrodesper square unit of surface area.

In order to make the formed electrodes independently chargeable, each ofthe electrode layers may be interconnected to routing layers ofconductors that terminate at a series of edge connection points. Anexternal electrical control system (not shown) may be connected to thoseconnection points and can be used to individually activate theelectrodes in any series and/or sequence as needed to move and/or dividethe liquid along the routing plate. In other words, in a manner similarto that described with reference to FIGS. 2 and 3, the charge of theelectrodes may be controlled so as to cause corresponding surfaceportions of the routing plate to exhibit hydrophobic characteristics orhydrophilic characteristics in order to divide and move liquid over therouting plate surface.

FIGS. 12A-12F show a plan view of the routing plate of FIG. 11 andexemplary steps for how liquid supplied to the routing plate 601 may bemoved along the filler rail 615 and arms 617 of the routing plate 601and divided into individual portions for dispensing from the nozzles ofa nozzle plate. The surface of the routing plate 601 illustrated inFIGS. 12A-12F is the surface in contact with the liquid and facing thenozzle plate.

With reference to FIG. 12A, liquid 700 to be dispensed is firstintroduced to the routing plate 601 into the main filler rail 615. Aswas described with reference to FIG. 10A, the liquid 700 may beintroduced, for example, via a port 640 in flow communication with thefiller rail 615, in a direction substantially perpendicular to the planeof the routing plate 601 (not shown in the figures). In a manner similarto that described with reference to FIG. 2A, the liquid 700 may beprevented from moving along the filler rail 615 by controlling theelectric charge of an array (e.g., a two-dimensional array) ofelectrodes underlying the filler rail 615 so as to make the surface ofthe filler rail 615 in contact with the liquid substantiallyhydrophobic, thereby repelling the liquid.

When it is desired to fill the filler rail 615, the electric charge ofan array of electrodes associated with the filler rail 615 may becontrolled so as to move the liquid 700 from the input port 640 andalong the rail 615. FIG. 12B shows an example of the liquid 700progressing from its input position in the upper corner of the routingplate 601 along the filler rail 615. FIG. 12C illustrates the mainfiller rail 615 being completely loaded with liquid 700.

Once the main filler rail 615 is completely loaded with liquid 700, asshown in FIG. 12C, the array of electrodes on the routing plate 601 mayagain be controlled so as divide the liquid in the filler rail 615 intoa plurality of smaller, individual portions that are substantiallyseparated from one another along the filler rail 615 and aligned withrespective arms 617. FIG. 12D illustrates this exemplary step, whereinthe electrodes of the routing plate 601 are selectively activated so asto partition the liquid 700 in the main filler rail 615 into a pluralityof separate portions that are in substantial alignment with theplurality of arms 617. In other words, electrodes corresponding tosurface portions of the filler rail 615 are selectively turned off so asto cause those surface portions to become hydrophobic, thus repellingthe liquid and partitioning the liquid into portions illustrated in FIG.12D. The partitioned portions of liquid in FIG. 12D collect over surfaceportions of the filler rail 615 corresponding to activated electrodes.The volume of liquid 700 in each of the partitioned portions in FIG. 12Dmay be substantially equal to the volume of each respective arm 617,such that each partitioned portion of liquid can substantially fill arespective arm 617.

An exemplary step of filling the arms 617 with the liquid 700 isillustrated in FIG. 12E. By selectively activating electrodes alignedalong the arms 617, the partitioned liquid in the filler rail 615 may bedrawn into the arms 617 and away from the filler rail 615. As the liquid700 progresses from the filler rail 615 into the arms 617, eventuallyall of the liquid 700 is drained from the filler rail 615 and iscontained in the arms 617, as illustrated in FIG. 12F. Thus, as shown inthe exemplary steps illustrated in FIGS. 12A-12F, liquid 700 may bemoved in a two-dimensional manner along the routing plate 601 byselectively controlling the electric field acting on the liquid andthereby controlling the wettability of surface portions in contact withthe liquid. Further, the liquid 700 may be divided into a plurality ofsmaller amounts corresponding to a plurality of substantially parallelrows corresponding to the arms 617.

Referring now to FIG. 12G, once the liquid 700 has been moved from thefiller rail 615 into the arms 617, various electrodes associated withthe arms 617 can be activated so as to again divide the liquid 700 inthe arms 617 into a plurality of individual portions 701 (for example onthe order of a few microliters or a few nanoliters, such as, forexample, ranging from about 0.01 microliters to about 100 microliters,for example about 1 microliter) aligned with a plurality ofpredetermined locations. In particular, according to an exemplaryaspect, the electrodes may be controlled so as to cause, viaelectrowetting, the liquid 700 in the arms to be divided into individualportions 701 located at predetermined locations on the routing plate 601that are aligned with respective openings leading to nozzles of a nozzleplate. FIG. 12G illustrates the exemplary step of dividing the liquid700 aligned with the arms 617 into a plurality of individual portions701 ready to be drawn into nozzles (e.g., into the reservoirs defined bythe inner surfaces of the nozzles) of a nozzle plate, such as, forexample, a nozzle plate like that shown in FIG. 10, for dispensing to atesting platform. It should be understood that the number and locationsof the individual portions 701 of liquid illustrated in FIG. 12G areexemplary and that the number, location, and volume of the individualportions 701 of liquid can be controlled as desired, for example, basedon the routing plate electrode configuration and/or the activation ofthe routing plate electrodes. The volume of individual portions ofliquid may vary depending on the application. For example, thedispensing devices disclosed herein could be configured to fill wellshaving a volume of about 10 nanoliters in a 24,000-well format. Inanother example, the dispensing device could be configured to fill wellshaving a volume ranging from about 10 microliters to about 100microliters in a 384-well format. It is envisioned that the dispensingdevices according to aspects of the invention could be configured todispense liquid in a relatively wide range of volumes.

As explained above, once the individual portions 701 have beenestablished, as shown in FIG. 12G, the electrodes associated with therouting plate 601 can again be controlled, e.g., via a controller (notshown) so as to cause the routing plate surface to become hydrophobic.At the same time, the electric potential of a nozzle plate associatedwith the routing plate 601 could be controlled so as to cause the nozzleinner surfaces to change from exhibiting hydrophobic characteristics toexhibiting hydrophilic characteristics, thereby drawing the individualportions 701 into the nozzles via electrowetting and/or capillarity.

Thus, the routing plate embodiment of FIG. 12 permits the suppliedliquid to be moved along in a somewhat bulk fashion, e.g., by volumescorresponding to the rail 615 and arms 617, prior to dividing the liquidinto individual portions desired for dispensing, such as, droplets, forexample. In this way, evaporation may be minimized and efficiencyimproved by eliminating the need to move individual droplets (or othersmall portions volumes desired to be dispensed) along relatively longroutes prior to dispensing the individual droplets.

A variety of techniques could be employed to fabricate a nozzle plateaccording to an exemplary embodiment. In an exemplary aspect, it isdesirable to fabricate the nozzle plate using a technique that permitsan ultra-high density nozzle configuration, such as that described inthe exemplary embodiment of FIGS. 13A and 13B. One such technique, forexample, could include a series of steps that are known to those skilledin the art of silicon micro-machining technologies. For example,through-holes could be formed in a silicon plate using a lithographicprocess followed by a deep reactive ion etching (DRIE) process. DRIE isa process that utilizes the pulsing of power during a reactive ion etchprocess to alternately etch a hole and then coat the side walls of thathole with polymeric byproducts that protect against etching. The processallows for the formation of structures having relatively high aspectratios. Once the through-holes have been formed, an atmosphericdown-stream plasma (ADP) process can be used in conjunction with astandard lithographic process to form the nozzles. ADP is an isotropicprocess which can lead to the tapered shape of the nozzles.

After formation of the nozzles, the inside surface of the nozzle plate(e.g., including the inside surface of the nozzles) can be coated with alayer of hydrophobic material, such as, for example, polymeric materialsand other resins that may be applied in solution where the solvent isallowed to evaporate leaving behind a film that may range from about 0.5micrometers to about 1.5 micrometers in thickness. Other film thicknessranges are envisioned and may be selected based on the particularapplication. Further, the nozzles may be coated with a layer ofconductive material and a layer of hydrophobic material in a mannersimilar to that described with reference to the wells 150 of FIG. 6.

Further information regarding DRIE can be found athttp://www.microfab.de/technologies/drie.htm, and further informationregarding ADP can be found at http://www.trusi.com/frames.asp.

A partial view of an exemplary embodiment of a nozzle plateconfiguration is shown in FIGS. 13A and 13B. It should be understoodthat for practical reasons the illustrations are partial views due tothe large number of nozzles that are envisioned for the exemplarybiological analysis applications described herein. For example, asexplained above, it may be possible to fabricate the nozzle plate suchthat there are as many as 400,000 nozzles arrayed across the surface ofan area approximating 3″×5″. Thus, only a small portion of a nozzleplate is illustrated in FIGS. 13A and 13B.

FIG. 13A illustrates a perspective view of a nozzle plate 900 viewedfrom the nozzle side 910, which is the side of the nozzle plate facingaway from the routing plate. As shown, the nozzle plate 900 includes aplurality of nozzles 920 arranged in an array of rows and columns. In anexemplary aspect, the nozzles 920 would be configured so as to alignwith predetermined locations on a testing platform, such as with wellson a titer plate, for example. FIG. 13B illustrates the opening side 915of the nozzle plate 900. The opening side 915 faces toward the routingplate and makes contact with the liquid to be dispensed. The openingside contains a plurality of openings 925 leading to the nozzles 920.

By way of example only, the nozzle plate 900 of FIGS. 13A and 13B mayhave an ultra-high density configuration and the nozzles 920 may bespaced from one another at approximately a 200 micrometer pitch. Theopenings of the nozzles from which the liquid is dispensed may have adiameter on the order of approximately 50 micrometers. It should beunderstood, however, that this nozzle plate configuration is exemplaryonly and that the number of nozzles, the nozzle dimensions, the spacingbetween the nozzles, and the nozzle arrangement may be chosen dependingon the dispensing application. For example, the volume of each nozzlemay be selected based on the desired volume of expressed liquid.

As with the embodiments of FIGS. 1-7, it is envisioned that routingplate/nozzle plate dispensing device discussed with reference to FIGS.9-13 could be configured as a stand-alone device, like a multi-tippipettor, for example, as a component of a device, like a liquidhandling mechanism within an instrument, or as part of an overall systemthat includes fluid handling between differing devices. Thus, as shownin FIG. 14, a routing plate/nozzle plate electrowetting dispensingdevice 1000 could replace the EWL 20 illustrated in FIG. 8. Thedispensing device 1000 could be manipulated and function as part of aworkstation system 11 in a manner similar to that described andillustrated in FIG. 8 and as described above with reference to thedescription of FIG. 8, with the exception that the EWL 20 of FIG. 8 isreplace with the routing plate/nozzle plate dispensing device 1000.

To the extent not already described above, it should be understood thatdevices and methods according to the invention may include variousexemplary aspects and/or features, some of which are set forth in thefollowing. A method for dispensing liquid may include aligning theliquid with a plurality of predetermined locations corresponding to atwo-dimensional array. Positioning the liquid may comprise moving theliquid, for example moving individual portions of liquid, in twodimensions. Dispensing aligned liquid may comprise moving the liquid,such as, for example, individual portions of liquid, in a directionsubstantially nonparallel to a plane defined by the two dimensions, forexample, in a direction substantially perpendicular to a plane definedby the two dimensions. A method for dispensing liquid may compriseproviding a first amount of liquid and dividing the first amount ofliquid into a plurality of individual portions less than the firstamount via electrowetting. Positioning liquid via electrowetting maycomprise positioning each of the plurality of individual portions intorespective alignment with each of the plurality of predeterminedlocations.

A method for dispensing liquid may comprise dividing the first amount ofliquid into a plurality of substantially parallel rows of liquid.Dividing the first amount of liquid may comprise dividing the firstamount of liquid into a plurality of droplets of liquid. Dividing mayfurther comprise dividing the first amount of liquid into a plurality ofindividual portions each ranging from about 0.01 microliters to about100 microliters, for example, from about 0.01 microliters to about 5microliters, for example about 1 microliter. A method for dispensingliquid may further comprise filling openings in a substrate with theliquid prior to dispensing the liquid through the openings. During thefilling, the openings may exhibit hydrophilic characteristics such thatthe liquid moves from predetermined locations into the openings.Further, prior to the filling, the openings may exhibit hydrophobiccharacteristics. Filling the openings with the liquid may comprisemoving the liquid into the openings via electrowetting. Dispensingaligned liquid through the plurality of openings in a substrate maycomprise dispensing the aligned liquid through a plurality of portopenings.

A method for dispensing liquid may further comprise dispensingindividual portions of liquid via a plurality of nozzle openings.Further, the method may comprise spotting the plurality of individualportions of liquid to a testing platform via the nozzle openings. Inanother aspect, dispensing the plurality of individual portions ofliquid may comprise dispensing the individual portions via a pluralityof port openings. The dispensing the plurality of individual portions ofliquid may include dispensing the plurality of individual portions ofliquid to a plurality of reservoirs. According to an aspect, thedispensing the plurality of individual portions of liquid to theplurality of reservoirs may comprise centrifuging the plurality ofindividual portions of liquid to the plurality of reservoirs. Theplurality of individual portions of liquid into may be dispensed to aplurality of reservoirs coated with a hydrophilic material.

According to another aspect, the dispensing the plurality of individualportions of liquid to the plurality of reservoirs may comprisedispensing the plurality of individual portions of liquid viaelectrowetting, for example, the dispensing may comprise dispensing theplurality of individual portions of liquid to a plurality of wells in atiter plate. The reservoirs may be coated with a layer of conductivematerial and a layer of hydrophobic material, and the dispensing viaelectrowetting may comprise altering an electric potential of thereservoir such that an inner surface of the reservoir exhibitshydrophilic characteristics.

According to yet a further aspect, the dispensing the individualportions of liquid to a plurality of reservoirs may comprise dispensingthe plurality of individual portions of liquid to the plurality ofreservoirs via capillarity, and the reservoirs may comprise hydrophiliccapillary tubes. In another aspect, the dispensing the plurality ofindividual portions of liquid to a plurality of reservoirs may comprisedispensing the plurality of individual portions of liquid viaelectrophoresis.

A method for dispensing liquid may further comprise moving alignedliquid to a plurality of reservoirs, wherein the moving comprisesaltering an electric potential of the reservoirs so as to cause an innersurface of the reservoirs to exhibit hydrophilic characteristics.

A device for positioning liquid to be dispensed may comprise, in anexemplary aspect, a first substrate and second substrate separated fromthe first substrate, the second substrate defining at least one openingtherethrough, the at least one opening being configured to permitpassage of an amount of liquid to be dispensed therethrough. The devicemay further comprise a chamber between the first substrate and thesecond substrate, the chamber being configured to contain liquid fordispensing. A controller may be configured to control an electric fieldacting on the liquid in the chamber so as to move a portion of theliquid in the chamber into alignment with the at least one opening andto dispense the portion of the liquid through the at least one opening.

According to additional exemplary aspects, the at least one opening maycomprise a plurality of openings. The at least one opening may alsocomprise an inner surface configured to exhibit hydrophobiccharacteristics prior to dispensing the liquid through the opening. Theinner surface of the at least one opening may be configured to exhibithydrophilic characteristics during the dispensing of the liquid throughthe opening. The second substrate may define a plurality of nozzles andthe plurality of openings may comprise nozzle openings. The plurality ofnozzles may be configured to dispense a plurality of individual portionsof the liquid to a titer plate. The controller may be configured tocontrol the electric field so as to move a plurality of individualportions of the liquid in the chamber into respective alignment with theplurality of openings. The controller may be further configured tocontrol the electric field so as to alter the wettability of at leastone surface portion in contact with the liquid in the chamber. Inanother aspect, the controller may be configured to control the electricfield so as to selectively alter the at least one surface portionbetween exhibiting hydrophobic characteristics and hydrophiliccharacteristics. The controller may be configured to selectively alterthe electric potential of the first and second substrates.

One or both of the first and second substrates may comprise ahydrophobic layer facing the chamber.

The device may further comprise a plurality of electrodes associatedwith the first substrate. The plurality of electrodes may beindependently electrically chargeable. The plurality of electrodes maybe disposed in an array of rows and columns. The at least one openingmay comprise a plurality of openings aligned with at least some of theplurality of electrodes, and the plurality of openings are configured tobe respectively aligned with a plurality of wells in a titer plate. Theplurality of electrodes may be configured to permit liquid supplied tothe chamber to be positioned so as to form a plurality of substantiallyparallel rows. The device may further comprise at least one additionalelectrode associated with the second substrate.

The device may further comprise a first substrate comprising adistribution channel configured to receive liquid to be dispensed. Thedistribution channel may be in flow communication with the plurality ofsubstantially parallel rows.

The device may further comprise an input port in flow communication withthe chamber.

In a further exemplary aspect a biological analysis system may comprisea device according to any exemplary aspects described above and aplurality of stations, including at least one liquid storing stationconfigured for storing liquid to be used in a biological analysisprocedure. The device may be configured to be movable between the atleast one liquid storing station and at least one other station of theplurality of stations.

The various dispensing devices and methods in accordance with aspects ofthe invention may allow for precise positioning of controlled smallvolumes (e.g., on the order of microliters or nanoliters) of liquid inorder to dispense the small volumes of liquid into specific formats on atesting platform. The various dispensing devices and methods could becontrolled via software in order to accommodate desired dispensingoperations. Further, it is envisioned that the dispensing devices andmethods disclosed herein could be produced in a cost-efficient manner,for example, by employing foundry services.

It is also envisioned that the exemplary devices and methods accordingto the invention could be used to perform multi-plexing procedures. Forexample, the devices could be provided with a plurality of input portsin which differing liquids could be input into the device alongseparated rows and/or columns of electrodes. By selectively activatingelectrodes, the input liquids could be kept segregated or combinedtogether as desired, and moved along the dispensing devices as desired.Reference is made to U.S. Publication No. 2003/0205632, incorporated byreference herein, for an exemplary method of how electrowettingprinciples can be utilized to perform multi-plexing procedures.

It should be understood that the particular electrode array that isutilized with the dispensing devices and methods described herein can beselected based on factors such as the desired movement of liquid throughthe dispensing device, the desired positioning of the individualportions of liquid to be dispensed, the desired amount of liquid in eachindividual portion which is to be dispensed, and other similar factors.

It should be noted that sizes and configurations of various structuralparts and materials used to make the above-mentioned parts areillustrative and exemplary only. One of ordinary skill in the art wouldrecognize that those sizes, configurations, and materials can be changedto produce different effects or desired characteristics. By way ofexample, it is envisioned that by utilizing lithographic materials andprocesses capable of micrometer sized features, the various dispensingdevices and methods according to aspects of the invention may be scaledto accommodate various volumes of liquids and portion densities (e.g.,high bandwith capability of the number of portions of liquid dispensedover a given area) as desired.

In this application, the use of the singular includes the plural unlessspecifically stated otherwise. It is noted that, as used in thisspecification and the appended claims, the singular forms “a,” “an,” and“the,” include plural referents unless expressly and unequivocallylimited to one referent. In this application, the use of “or” means“and/or” unless stated otherwise. Furthermore, the use of the term“including”, as well as other forms, such as “includes” and “included”,is not limiting. As used herein, the term “include” and its grammaticalvariants are intended to be non-limiting, such that recitation of itemsin a list is not to the exclusion of other like items that can besubstituted or added to the listed items. Also, terms such as “element”or “component” encompass both elements and components comprising oneunit and elements and components that comprise more than one subunitunless specifically stated otherwise. Wherever possible, the samereference numbers will be used throughout the drawings to refer to thesame or like parts.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the described subject matter inany way. All literature and similar materials cited in this application,including patents, patent applications, articles, books, treatises, andinternet web pages are expressly incorporated by reference in theirentirety for any purpose. In the event that one or more of theincorporated literature and similar materials defines or uses a term insuch a way that it contradicts that term's definition in thisapplication, this application controls. While the present teachings aredescribed in conjunction with various exemplary embodiments, it is notintended that the present teachings be limited to such embodiments. Onthe contrary, the present teachings encompass various alternatives,modifications, and equivalents, as will be appreciated by those of skillin the art.

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing quantities, percentages orproportions, and other numerical values used in the specification andclaims, are to be understood as being modified in all instances by theterm “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in the following specification andattached claims are approximations that may vary depending upon thedesired properties sought to be obtained by the present invention. Atthe very least, and not as an attempt to limit the application of thedoctrine of equivalents to the scope of the claims, each numericalparameter should at least be construed in light of the number ofreported significant digits and by applying ordinary roundingtechniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Moreover, all ranges disclosed hereinare to be understood to encompass any and all subranges subsumedtherein. For example, a range of “less than 10” includes any and allsubranges between (and including) the minimum value of zero and themaximum value of 10, that is, any and all subranges having a minimumvalue of equal to or greater than zero and a maximum value of equal toor less than 10, e.g., 1 to 5.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the structure andmethodology of the present invention. Thus, it should be understood thatthe invention is not limited to the examples discussed in thespecification. Rather, the present invention is intended to covermodifications and variations. Other embodiments of the invention will beapparent to those skilled in the art from consideration of thespecification and practice of the invention disclosed herein.

What is claimed is:
 1. A method for dispensing liquid for use inbiological analysis, the method comprising: filling a hydrophilic inputport with a volume of liquid; aliquoting a portion of the volume ofliquid in the input port via electrowetting; positioning the aliquot viaelectrowetting, the positioning comprising aligning the aliquot with oneof a plurality of predetermined locations; dispensing the alignedaliquot from the plurality of predetermined locations through aplurality of openings respectively aligned with the predeterminedlocations; and repeating the steps of aliquoting, positioning anddispensing until the volume of liquid in the input port has beendispensed.
 2. The method of claim 1, further comprising dividing theliquid into a plurality of individual portions via electrowetting,wherein the positioning comprises aligning each of the plurality ofindividual portions with the plurality of predetermined locations. 3.The method of claim 1, wherein the dispensing the aligned liquidcomprises dispensing the aligned liquid through a plurality of openingsof a plurality of nozzles.
 4. The method of claim 3, further comprisingspotting the liquid to a testing platform via the plurality of nozzles.5. The method of claim 1, wherein the dispensing the aligned liquidincludes dispensing the liquid through the plurality of openings to aplurality of reservoirs.
 6. The method of claim 5, wherein thedispensing the aligned liquid to the plurality of reservoirs comprisescentrifuging the liquid into the reservoirs.
 7. The method of claim 5,wherein the dispensing the aligned liquid to a plurality of reservoirscomprises dispensing the aligned liquid to a plurality of reservoirscoated with a hydrophilic material.
 8. The method of claim 5, whereinthe dispensing the aligned liquid to a plurality of reservoirs comprisesdispensing the aligned liquid to the plurality of reservoirs viaelectrowetting.
 9. The method of claim 8, wherein the dispensing thealigned liquid to the plurality of reservoirs comprises dispensing thealigned liquid to a plurality of reservoirs coated with a layer ofelectrically conductive material and a layer of hydrophobic material.10. The method of claim 8, wherein the dispensing the liquid to aplurality of reservoirs via electrowetting comprises dispensing theliquid to a plurality of wells in a titer plate via electrowetting. 11.The method of claim 5, wherein the dispensing the liquid to a pluralityof reservoirs comprises dispensing the liquid via capillarity.
 12. Themethod of claim 11, wherein the reservoirs comprise hydrophiliccapillary tubes.
 13. The method of claim 11, wherein the dispensing theliquid to a plurality of reservoirs comprises dispensing the liquid viaelectrophoresis.
 14. The method of claim 5, wherein the dispensing theliquid to a plurality of reservoirs comprises dispensing the liquid to aplurality of wells in a titer plate.
 15. A method for dispensing liquidfor use in biological analysis, the method comprising: supplying aliquid to be dispensed to a housing comprising an interior surface;altering a wettability of the interior surface so as to divide theliquid in the housing into a plurality of individual portions of liquidand to move the plurality of individual portions of liquid to aplurality of respective predetermined locations; dispensing theplurality of individual portions of liquid from the plurality ofpredetermined locations through a plurality of openings respectivelyaligned with a plurality of wells on a testing device clamped to thehousing.
 16. The method of claim 15, wherein the interior surfacecomprises an electrical conductor.
 17. The method of claim 16, whereinthe interior surface comprises a dielectric covering an electricalconductor.
 18. A method for dispensing liquid for use in biologicalanalysis, the method comprising: filling a hydrophilic input port with avolume of liquid; dividing the volume of liquid in the input port into aplurality of individual portion via electrowetting; moving the pluralityof individual portions via electrowetting so as to align the individualportions with a plurality of predetermined locations; and moving thealigned liquid via electrowetting from the plurality of predeterminedlocations to a plurality of reservoirs aligned with the predeterminedlocations.
 19. The method of claim 18, wherein the moving the alignedliquid to a plurality of reservoirs comprises moving the aligned liquidto a plurality of nozzle reservoirs.
 20. The method of claim 19, furthercomprising dispensing the liquid from the plurality of nozzle reservoirsto a plurality of locations on a testing platform.